Pairwise detection of site-specific receptor phosphorylations using single-molecule blotting

Kyung Lock Kim, Daehyung Kim, Seongsil Lee, Su-Jeong Kim, Jung Eun Noh, Joung-Hun Kim, Young Chan Chae, Jong-Bong Lee and Sung Ho Ryu ()
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Kyung Lock Kim: Pohang University of Science and Technology (POSTECH)
Daehyung Kim: Pohang University of Science and Technology (POSTECH)
Seongsil Lee: School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH)
Su-Jeong Kim: Pohang University of Science and Technology (POSTECH)
Jung Eun Noh: Pohang University of Science and Technology (POSTECH)
Joung-Hun Kim: Pohang University of Science and Technology (POSTECH)
Young Chan Chae: Pohang University of Science and Technology (POSTECH)
Jong-Bong Lee: Pohang University of Science and Technology (POSTECH)
Sung Ho Ryu: Pohang University of Science and Technology (POSTECH)

Nature Communications, 2016, vol. 7, issue 1, 1-10

Abstract: Abstract Post-translational modifications (PTMs) of receptor tyrosine kinases (RTKs) at the plasma membrane (PM) determine the signal transduction efficacy alone and in combination. However, current approaches to identify PTMs provide ensemble results, inherently overlooking combinatorial PTMs in a single polypeptide molecule. Here, we describe a single-molecule blotting (SiMBlot) assay that combines biotinylation of cell surface receptors with single-molecule fluorescence microscopy. This method enables quantitative measurement of the phosphorylation status of individual membrane receptor molecules and colocalization analysis of multiple immunofluorescence signals to directly visualize pairwise site-specific phosphorylation patterns at the single-molecule level. Strikingly, application of SiMBlot to study ligand-dependent epidermal growth factor receptor (EGFR) phosphorylation, which is widely thought to be multi-phosphorylated, reveals that EGFR on cell membranes is hardly multi-phosphorylated, unlike in vitro autophosphorylated EGFR. Therefore, we expect SiMBlot to aid understanding of vast combinatorial PTM patterns, which are concealed in ensemble methods, and to broaden knowledge of RTK signaling.

Date: 2016
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DOI: 10.1038/ncomms11107

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